Analytical apparatus

ABSTRACT

A surface plasmon resonance apparatus for detecting a soluble analyte (e.g. a protein) or a particulate analyte (e.g. a cell), the apparatus comprising: (a) a sensor block adapted to receive a sensor, said sensor, for example a sensor slide, having a metallized sensor surface capable of binding the analyte; (b) a light source capable of generating an evanescent wave at the sensor surface of a sensor slide on the sensor block; (c) a first detector capable of detecting light from the light source which is internally reflected from the sensor surface; and (d) a second detector (e.g. a video camera) capable of detecting light scattered or emitted from an analyte bound thereto. Optionally the apparatus further comprises a second light source for increasing the intensity of the light scattered or emitted from an analyte bound to the sensor surface, preferably, this is sited to such as to minimize the amount of light transmitted therefrom which is detected by the first detector. Also disclosed are sensors adapted for use in the apparatus, and methods of detecting analytes in samples comprising exposing samples to the sensor surface of the apparatus.

This application is a division of application Ser. No. 09/308,501, filedMay 7, 1999, now U.S. Pat. No. 6,268,125 the entire content of which ishereby incorporated by reference in this application and which is a 371of PCT GB97/03037 filed Nov. 5, 1997.

TECHNICAL FIELD

The present invention relates broadly to apparatus for the detection ofanalytes. The invention further relates to methods employing suchapparatus.

BACKGROUND ART

The use of Surface Plasmon Resonance (SPR) for the detection of smallsoluble analytes from solution is well known (see e.g. “Advances inBiosensors—A Research Annual Vol 1. 1991” Ed. A P F Turner, Pub. JaiPress Ltd, London).

Briefly, an SPR apparatus generally comprises a light source forgenerating polarised light; a sensor, the outside of which is metalcoated and may be contacted with a sample solution, and means fordetecting the light which is internally reflected from the inner sensorsurface.

In the absence of bound analyte, light is totally internally reflectedat an incident angle characteristic of the refractive index (RI) of thesensor and of the sample solution. At a particular incident angle (the‘SPR angle’), interaction of the metal with the evanescent wave set upby internal reflection of the polarised light causes a drop in intensityof the reflected light. This drop can be observed using the lightdetector.

The binding of analyte to the sensor surface, within the evanescent wavezone, alters the RI of the sensor and this perturbs the SPR angle. Thisperturbation can be observed using the light sensor and related to thesurface concentration of analyte.

SPR detection in the literature has generally been limited to use withsoluble molecular size analytes e.g. biomolecules such as proteins andnucleic acids which are specifically bound within the evanescent zoneusing appropriate ligands.

However, the SPR apparatus in the art to date has not been suitable foraccurately detecting sample materials with both soluble and insolubleanalytes therein. In particular, due to the more limited way in which(for instance) roughly spherical cells of several μm diameter interactwith the evanescent zone, only fairly high concentrations (e.g.10⁷-10⁸/ml) have been detectable using SPR. Thus in order to detectcells, as opposed to (for instance) protein antigens, further apparatus,and hence more cost, time and experimentation, have been required. Forinstance cells have frequently been detected using culture techniquesfollowed by specific detection.

DISCLOSURE OF THE INVENTION

1. A surface plasmon resonance apparatus for detecting singleparticulate analytes, the apparatus comprising:

(a) a sensor, or means to receive a sensor, said sensor providing ametallised surface capable of binding the analyte;

(b) a light source capable of generating an evanescent wave at thesensor surface;

(c) a detector capable of detecting light scatterered or emitted from asingle particulate analyte bound at the sensor surface, said detectorbeing located on the opposite side of the sensor surface to which lightfrom said source is incident.

Suitable sensors are slides.

Possible analytes may include those particulate or insoluble analytescontaining or consisting of biomolecules, for instance bacteria or othercells, spores, viruses or virions etc., or biomolecules themselves suchas proteins or polynucleotides. Possible bacterial targets includecryptosporidium, E. coli, salmonella etc.

The apparatus may thus be used with a wide variety of samples suspectedor known to contain analytes. For examples environmental samples such aswater, or biological samples.

Broadly speaking the apparatus operates as follows: in use the seconddetector detects the binding of soluble analytes to the sensor surfaceby detecting the changes in the intensity of light internally reflectedfrom the sensor surface, whereas the first detector detects the bindingof particulate analytes to the sensor surface by detecting the lightscattered or emitted from the analytes bound thereto. The apparatus ofthe present invention is therefore capable of the sensitive detection ofboth soluble and particulate analytes, and thus may provide a quicker,cheaper or more sensitive alternative to the methods and apparatuspresently used in the art.

It is important to stress the different functions of the detectors inthe apparatus. The second detector must be arranged to detect lightinternally reflected from the sensor surface, the intensity of thislight being dependent on the SPR effects occurring as analytes(especially soluble ones) bind at the sensor surface altering therefractive index of the sensor/sample interface. The detector may be a2-D array detector as described in more detail in the Examples below.

By contrast the fist detector detects light which is scattered orotherwise emitted (optionally by fluorescence) from analytes (especiallyparticulate ones) which interact with the evanescent field at the sensorsurface. This may give a sensitivity for detecting large particulateanalytes several orders of magnitude higher than would be obtainableusing pure SPR. Clearly the nature of the first detector used willdetermine the sensitivity and acuity of the detection, but in preferredembodiments single cells bound within the evanescence zone may bedetected and resolved using the first detector while the bulk bindingeffects of soluble molecules may be detected using the first.

Preferably the first detector is a video camera (e.g. a Charge CoupledDetector [CCD] camera), but any kind of light detector appropriated fordetecting light scattered or emitted from the analytes may be used e.g.a 2-D diode array, a photomultiplier etc.

In one embodiment the first detector is located on the same side of thesurface as the light source such as to be capable of detecting lightwhich is back-scattered or emitted when an analyte is bound to thereto.

The term ‘light source’ as used herein means any source of lightradiation, including where appropriate the tip of an optical fibre whichis attached to a remote radiation source.

In a different embodiment, the first detector is located on the oppositeside of the surface as the light source detector such as to be capableof detecting light which is scattered or emitted when an analyte isbound to thereto.

In either case it may be desirable that the first detector is locatedsuch as to be capable of detecting light scattered or emitted at apredetermined angle, for example substantially normally, to the sensorsurface. This will minimise interference from light which is beingtotally internally reflected from the surface.

Generally the sensor block will comprise a prism or a hemicylinder, suchas are known to those skilled in the art of SPR detection. The sensorblock is adapted to receive the detachable sensor which provides themetallised surface. The adaptation may simply consist of providing ageneral area to mount the sensor such as a slide, or the block may bespecially shaped or configured to receive it e.g. in a groove orproperly-dimensioned well.

The block and or sensor may in addition be adapted to form all or partof one wall of a flow channel, through which a liquid sample can flow inliquid contact with the metallised surface. An apparatus comprising sucha flow channel forms one embodiment of the first aspect of theinvention.

Preferably the metallised sensor surface is adapted or otherwisefunctionalised such as to facilitate the immobilisation ofmacromolecules which are capable of specifically binding biomoleculesthereto. For instance the sensor may have a hydrophilic dextran surface.Antibodies may then be immobilised thereto in order to specifically bindantigenic analytes. Alternatively a polynucleotide probe may beimmobilised for specifically binding a polynucleotide analytes.

Preferably the e.g. antibodies are bound only to discrete portions ofsurface in order to facilitate the detecting light which is scattered oremitted when an analyte is bound to thereto. These portions may then bevisualised (and possibly further resolved) by the second detector ascontrasting discrete bright areas against the darker portions of thesurface which do not have macromolecules bound to them.

The surface may have greater then one type of macromolecule immobilisedthereto for specifically binding greater then one type of antigen. Thedifferent types of e.g. antibody may be bound in known discrete areas inorder to easily identify which antigen is being specifically bound.

In one further embodiment of the invention, the apparatus includes asecond light source. This can be used to increase the intensity of thelight scattered or emitted from the sensor surface when an analyte isbound thereto. Although this embodiment requires additional components,it has the advantage that the light source can be optimised (e.g.wavelength, angle of incidence against the sensor surface, intensity)for light scattering and/or fluorescence.

It may be desirable to locate the second light source such as tominimise the amount of stray light emitted therefrom which is detectedby the second detector.

This may be done by locating the second light source such that lightemitted therefrom travels along the same light path but in the oppositedirection from the light from the first light source which is internallyreflected from the sensor surface to the second detector, as is shown inthe Figures below.

The light source(s) used can be selected without undue burden by thoseskilled in the art. In order to maximise intensity, and hencesensitivity, the or each light source may be a laser light source, or alight emitting diode.

In a second aspect of the invention there is disclosed a method ofdetecting an analyte in a sample comprising exposing the sensor surfaceof an apparatus as described above to the sample. The analyte may thenbe detected by the first or second detector.

For instance a soluble analyte in a sample may be detected by detectingthe changes in the intensity of light internally reflected from thesensor surface. A particulate analyte in a sample may be detected bydetecting the light scattered or emitted from the analytes bound to thesensor surface. Preferably the apparatus is arranged such that solubleor particulate analytes may be detected simultaneously.

The means adapted to secure the second detector may comprise a holder orclamp positioned and/or dimensioned to receive e.g. a video camera andassociated optics, such that it can detect light scattered or emittedfrom the sensor surface. The holder or clamp may be moveable in apre-determined way to facilitate the function of the second detectorwhen in place e.g. to allow focusing.

Preferably the means are adapted to secure the first detector such thatit is capable of detecting light emitted at a predetermined angle, forexample substantially normally, to the sensor surface.

The second detector of the apparatus may also be adapted such as toreceive a second light source. The adaptation may be such that thesecond light source, when in place, is configured to minimiseinterference with the second detector by being directed away from it, asdescribed above.

A fifth aspect is a sensor having a metallised surface and being adaptedfor the apparatus above, in particular so as to allow light emitted orscattered from the sensor surface to be transmitted to the firstdetector. The sensor may comprise a slide and the surface may befunctionalised in discrete sections as described above.

While both SPR and particle detection by optical means are known in theprior art, the idea of modifying SPR apparatus (and, in particular,exploiting the existing light source and analyte binding site) to allowsimultaneous detection of soluble and particulate analyte is a usefulinvention that is not obvious.

FIGURES

FIG. 1 Shows a schematic diagram of a surface plasmon resonanceapparatus for detecting a soluble or a particulate analyte, as describedin more detail in Example 1.

FIG. 2 Shows a block diagram of the complete instrument of Example 1.

FIG. 3 Shows how the apparatus may be used to detect multiple analytes.

FIGS. 3(a) and (b) show the light source, hemicylinder (plus detectionsurface), and CCD array detector schematically.

FIG. 3(c) shows a detail of the CCD array.

FIG. 4 Shows bound particles scattering light from the metalliseddetection surface of a hemicylinder sensor. The light can be detected bya video camera (not shown).

FIG. 5 shows scattering from bacterial particles above a silver surface:the points of light represent scattered light from Erwinia herbicola.

EXAMPLES Example 1 Surface Plasmon Resonance Apparatus for Detecting aSoluble or a Particulate Analyte

FIG. 1 Shows a schematic diagram of a surface plasmon resonanceapparatus for detecting a soluble or a particulate analyte, such ascould be constructed (in the light of the present disclosure) by thoseskilled in the art. A block diagram of the components of the apparatusis shown in FIG. 2.

This system may be rearranged if desired, for instance the polariser maybe placed after the hemicylinder if required.

Considering FIG. 1, the light path to the first detector (‘CCD Array’)is from the light source at the left, through the beam splitter (whichsplits a portion to the reference detector), through a polariser andfocusing lens, off the internal surface of the hemicylinder, through acollimating lens and into the CCD array.

The light path is shown schematically in FIG. 3(a). An extendedcollimated source may be used to illuminate the hemicylinder surfacecontinuously over a range of incident angles, as shown in FIG. 3(b). TheCCD array is composed of a pixelated array of individual light sensors,each detecting a different reflected angle or being used to detect adifferent sample analyte (in this case 4 different samples) as shown inFIG. 3(c). This allows the rapid monitoring without moving parts.

Considering FIG. 1, the light path to the second detector (‘CCD camera’)is from the light source at the left, through the beam splitter (whichsplits a portion to the reference detector), through a polariser andfocusing lens and onto the hemicylinder.

The intensity is supplemented in this embodiment by light from thevisible laser diode on the right which travels away from the CCD arrayand through the collimating lens on the right and onto the hemicylinder.The evanescent field generated on the upper, metallised, surface of thehemicylinder causes particles bound therein to scatter light as depictedin FIG. 4. The scattered light is focused through a lens and detected bythe CCD camera.

Naturally if the particles were fluorescently labelled, using reagents(e.g. fluourescein) and methods well known to those skilled in the art,then the CCD camera could detect emitted light as the particles areexcited by the evanescent field.

Devices according to Example 1 may be constructed based on existing SPRmachines but having the additional components described above. Themachines and components may be those available commercially. Forinstance the light source may advantageously be an edge emitting LED asused in fibre-optic communications (e.g. EG&G type S86018). A stabilisedpower supply may be used to minimise artefacts.

The sensor may be metal-coated microscope slide (or similar thicknessdielectric) which is index matched onto the hemicylinder with fluid ofsimilar refractive index. A portion of the hemicylinder may be groundoff to accommodate the slide.

The CCD array (with ‘pixels’ about 20 μm²) may be of a type developedfor video use. Readout from CCD was accomplished by transferring asample-area row to a readout or row register. Correlated Double Sampling(CDS) may be used to eliminate noise. The analog output can be passed toa digital signal processor via an ADC. A suitable processor is an AnalogDevices ADSP-2105. This can communicate with an external host PC via abi-directional parallel port.

The CCD video camera can be a conventional, commercially available, onee.g. as sold by Hamamatsu (Japan).

Example 2 Method of Use of Surface Plasmon Resonance Apparatus

In use, in order to correct for differences in source intensity alongthe collimated beam, a calibration can be carried out before theexperiment. The sensor surface is then exposed to the sample(s). Thehost selects monitoring angles through using reflectivity vs. anglescans. Data is then acquired over a set time period and displayed by thehost PC.

Example 3 Detection of Particulate Analyte Using the Second Detector

In order to illustrate the light scattering technique, a glassmicroscope slide was coated with silver for optimum surface plasmonresonance (48 nm). The slide was then mounted onto a glasshemicylindrical prism and illuminated with a 3 mW helium-neon laser. Theslide was covered with a film of bacteria (Erwinia herbicola) at1×10⁶/ml in phosphate buffered saline solution. The bacteria were thenallowed to adsorb onto the surface of the silver microscope slide.

The bacteria were then allowed to adsorb onto the surface of the silvermicroscope slide. The output from the CCD array above the SPR surface isa normal video output with 256 levels of brightness. Observation abovethe silver surface showed that initially all pixels on the CCD cameragave a low reading (1-20) and the surface appeared dark. As the bacteriaapproached the surface, the brightness increased for those pixelsspecifically aligned with the areas where the bacteria were on thesurface. The maximum brightness level recorded from the light scatteredby the bacteria at the surface was 230. The appearance of the surfacewas that of a dark background with bright spots associated with thebacteria on the surface (See FIG. 5).

As a control, a film of phosphate buffered saline without bacteria wasused to cover the silver surface of a similar microscope slide. Thistime, no scattering from the surface was observed.

What is claimed is:
 1. A surface plasmon resonance apparatus fordetecting a soluble and, or a particulate analyte, the apparatuscomprising: a sensor providing a metallised surface capable of bindingthe analyte; a light source capable of generating an evanescent wave atthe sensor surface; a first detector comprising means for producing animage of the metallised surface derived from light scattered by analyteparticles bound thereto and a second detector capable of detecting lightfrom the light source which is internally reflected from the metallisedsurface.
 2. Apparatus according to claim 1 where the first detector is avideo camera.
 3. Apparatus according to claim 1 where the first detectoris a 2-dimensional diode array.
 4. A method of detecting particulateanalyte comprising: (a) binding the analyte to a sensor, said sensorcomprising a metallised surface capable of binding the analyte; (b)providing a light source capable of generating an evanescent wave at thesensor surface; (c) imaging the metallised surface by means of lightscattered by analyte particles bound to the metallised surface and (d)detecting light from the light source which light is internallyreflected from the metallised surface.